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OPN5 / Opsin-5 · IHC design guide
Design Immunohistochemistry for OPN5
Plan chromogenic IHC of paraffin sections with retinal photoreceptors as a positive reference (HPA tissue IHC). This guide covers fixation consistency, the catalog antibody’s 2–5 μg/ml IHC range (datasheet A08880-2), and interpretation of membrane staining (UniProt).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
OPN5 Immunohistochemistry Experimental Design Guide
Expected localisation, validated protocols, controls and antibodies — the at-a-glance facts below, then the full design guide.
Must know before staining
| Expected localisation | Membrane expected (UniProt); retinal photoreceptors stain (HPA tissue IHC) | |
| Staining pattern | Retinal photoreceptor cells and brain processes; compartment unspecified (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A08880-2) | |
| Positive control | Retina | |
| Negative control | Adipose tissue+4 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep paraffin section fixation consistent (standard IHC practice; not target-specific); Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A08880-2) | |
| Caveat | Testis-enriched RNA may mislead tissue choice; retinal staining is high (HPA tissue IHC) | |
| Regulation | Corneal injury induction, by similarity (UniProt) | |
| Isoform / epitope | No isoforms annotated; epitope side matters across 7 TM spans (UniProt) |
Section 1
Recommended OPN5 IHC & IF Protocols
Start with the catalog antibody’s EDTA pH 8.0 heat retrieval (datasheet: A08880-2), then compare the published duck and gander hypothalamus IHC protocols (PMC10964073; PMC11758404).
| Sample | Paraffin-embedded human liver cancer tissue; fixative not specified (datasheet A08880-2) |
| Fixation | Image fixative and duration unreported (datasheet A08880-2); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat retrieval: EDTA pH 8.0 (datasheet A08880-2); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A08880-2) |
| Primary antibody | Rabbit anti-OPN5, 2-5 μg/ml (datasheet A08880-2) |
| Primary incubation | Overnight at 4 °C (datasheet A08880-2) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet A08880-2) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | OPN5-positive staining in photoreceptor cells of retina (HPA tissue IHC: High). HPA tissue profile: Expression in photoreceptor cells in retina and processes in brain. No signal in the no-primary control. |
💡Decision noteTry EDTA pH 8.0 heat retrieval first (datasheet: A08880-2); the gander study reports antigen repair without specifying its method (PMC11758404).
Section 2
What Is the Expected OPN5 Staining Pattern?
OPN5 is a seven-pass cell-membrane protein (UniProt Q6U736 topology and subcellular location). In paraffin-section IHC, expect high staining in retinal photoreceptor cells and staining in brain processes (HPA tissue IHC). The HPA tissue pattern is rated Supported by external characterization; that rating does not establish the exact membrane domain stained within a photoreceptor (HPA tissue IHC; UniProt Q6U736 topology).
| Photoreceptor cells show strong, cell-associated staining in retina, with a membrane-like distribution. | This matches the reported high photoreceptor-cell signal and OPN5 membrane location (HPA tissue IHC; UniProt Q6U736). Record the retinal layer and cellular distribution; HPA does not specify a positive photoreceptor subcompartment (HPA tissue IHC). |
| Staining follows processes in brain sections. | Process staining fits the HPA tissue profile; UniProt also reports OPN5 detection in brain (HPA tissue IHC; UniProt Q6U736 tissue specificity). Interpret a process pattern in its anatomical context rather than treating every stained brain cell as a documented positive cell type (HPA tissue IHC). |
| A strong, isolated nuclear pattern dominates the retinal signal. | Nuclear-only staining conflicts with the reported cell-membrane location and seven-pass topology (UniProt Q6U736). Treat it as suspect, then compare its distribution with the positive retinal pattern and a no-primary control before assigning it to OPN5 (HPA tissue IHC; general IHC practice). |
| Cells reported as unstained show convincing chromogen, such as adipocytes in adipose tissue or glandular cells in adrenal gland. | Those cell types are listed as not detected in HPA tissue IHC. Widespread signal there raises concern for nonspecific antibody binding or endogenous detection activity; a negative HPA result is a reference pattern, not proof of absolute protein absence (HPA tissue IHC; general IHC practice). |
| The whole section has diffuse staining, or retinal photoreceptor cells have no discernible signal. | Diffuse staining obscures the cell-specific pattern, while no signal in a known-positive retina prevents interpretation of a negative test section (HPA tissue IHC; general IHC practice). First establish whether the positive control has selective photoreceptor staining before scoring other tissue (HPA tissue IHC; general IHC practice). |
💡Expected OPN5 appearanceCall the result positive when retinal photoreceptor cells show high, cell-associated staining consistent with a membrane protein; diffuse section-wide color, isolated nuclear color, or strong staining in HPA-negative cell types is suspect (HPA tissue IHC; UniProt Q6U736; general IHC practice).
| Topology and epitope location | OPN5 has 7 transmembrane segments and extracellular and cytoplasmic regions (UniProt Q6U736 topology). Interpret membrane-like signal accordingly; the supplied record gives no antibody epitope, so it cannot predict which face an antibody recognizes or a target-specific retrieval requirement (UniProt Q6U736; supplied antibody record). |
| Protein processing and variants | UniProt lists one chain spanning residues 1–354, no signal peptide or propeptide, and 0 annotated isoforms (UniProt Q6U736). These annotations give no basis for assigning a separate staining pattern to cleavage or an alternate isoform; they do not prove that every tissue shares identical protein processing (UniProt Q6U736). |
| Tissue evidence and antibody validation | HPA rates retinal photoreceptor staining High and the tissue IHC result Supported; the listed antibody, HPA026084, is also IHC Supported (HPA tissue IHC; HPA antibodies). HPA reports testis-enriched RNA, which alone does not establish a positive testis protein-staining pattern (HPA tissue IHC). |
| Can ICC/IF confirm this IHC pattern? | HPA summarizes the subcellular location as membrane but provides no ICC/IF images, and ICC validation is unlisted for HPA026084 (HPA subcellular; HPA antibodies). Treat any IF result as a separate observation requiring its own controls, without inferring an established ICC staining pattern from the IHC evidence (HPA subcellular; HPA antibodies; general IF practice). |
| Situation | Likely cause | Next action |
|---|---|---|
| Retina positive control has no photoreceptor signal. | A missing expected signal may reflect a failed IHC run or unsuitable assay settings; the cause cannot be identified from the absence alone (HPA tissue IHC; general IHC practice). | Target-specific fixation effects are not established by the supplied assay evidence. Verify with a matched IHC source before attributing a result to fixation. |
| Most cells or extracellular spaces show similar brown color. | Section-wide color is inconsistent with the reported cell-selective photoreceptor pattern and may reflect background or endogenous detection activity (HPA tissue IHC; general IHC practice). | Compare with a no-primary control and assess detection-system blocking and washes; score OPN5 only where cellular localization remains distinguishable (general IHC practice). |
| Signal is strongest in HPA-negative reference cells. | Adipose adipocytes and adrenal glandular cells are reported not detected; strong signal there may indicate nonspecific binding or endogenous activity (HPA tissue IHC; general IHC practice). | Run a matched negative reference section and no-primary control, then compare their color distribution with retina before calling the test section positive (HPA tissue IHC; general IHC practice). |
| Predominantly nuclear staining appears in retina. | A nuclear-only pattern does not fit UniProt's cell-membrane assignment or seven-pass topology (UniProt Q6U736). | Review the counterstain and no-primary control, and compare nuclear color with cell-associated photoreceptor signal in the positive control (HPA tissue IHC; general IHC practice). |
| Brain staining is present but its cellular source is unclear. | HPA describes staining in brain processes without naming their cell of origin; UniProt reports detection in brain without assigning every stained process to a cell type (HPA tissue IHC; UniProt Q6U736 tissue specificity). | Report the anatomical site and process-like distribution seen on the slide; defer a specific cell-type claim until independently supported (HPA tissue IHC; general IHC practice). |
| A second section differs in intensity from the positive retina. | HPA's High rating applies to retinal photoreceptor cells, not to all tissues or preparations (HPA tissue IHC). | Compare sections within the same controlled IHC run and score location and cell type before intensity; do not transfer the retinal intensity threshold to an uncharacterized tissue (HPA tissue IHC; general IHC practice). |
Experiment evidence
IHC & IF images showing OPN5 staining
A08880-2 · IHCIHC analysis of OPN5 using anti-OPN5 antibody (A08880-2). OPN5 was detected in a paraffin-embedded section of human liver cancer tissue. Hea
A08880-2 · IHCIHC analysis of OPN5 using anti-OPN5 antibody (A08880-2). OPN5 was detected in a paraffin-embedded section of human liver tissue. Heat media
A08880-2 · IFIF analysis of OPN5 using anti-OPN5 antibody (A08880-2). OPN5 was detected in a paraffin-embedded section of human liver cancer tissue. Heat
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000124818-OPN5/tissue/cerebral+cortex#imid_20110645
HPA · RetinaHuman Protein Atlas IHC, Retina. Source: https://www.proteinatlas.org/ENSG00000124818-OPN5/tissue/retina#imid_845
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000124818-OPN5/tissue/colon#imid_20110571
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000124818-OPN5/tissue/liver#imid_20110588
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for OPN5 IHC & IF
🧪Run retina first: photoreceptor cells should stain (HPA: High in retinal photoreceptor cells). Use adipose tissue adipocytes as a negative comparator (HPA: Not detected in adipocytes); on the retina slide, assess other cell types as internal negative candidates only after confirming their staining status, since the supplied HPA row identifies photoreceptors alone.
Positive control tissue: Retina (Photoreceptor cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA carries no ICC-IF cell line for OPN5; derive a cell-line control from the positive tissue's cell type (Photoreceptor cells) and confirm it by RNA or western blot first.
Technical controls: Include no-primary (secondary-only), host- and clonality-matched rabbit IgG isotype, and OPN5-knockout tissue if available (caption: rabbit primary; standard IHC control practice). Block endogenous peroxidase and distinguish retinal pigment from DAB signal (standard chromogenic IHC practice).
⚠️Feasibility: No target-specific fixation window or fixation effect is reported in the supplied evidence; the selected A08880-2 paraffin-section caption does not state a fixative (selected-SKU caption: fixative unreported). The caption uses heat retrieval in EDTA at pH 8.0, but does not establish that retrieval is required or that frozen sections or IF are easier (selected-SKU caption). Retinal pigment can complicate chromogenic scoring, so interpret photoreceptor staining against the negative controls (standard retinal IHC practice).
HPA tissue IHC evidence for OPN5
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Supported — External characterization data supports antibody staining.). Rows are taken directly from the HPA tissue chart — click any row's HPA link to view the source.
Positive expression · recommended positive controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Retina | Photoreceptor cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Adrenal gland | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Appendix | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Breast | Adipocytes | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced OPN5 IHC Tips
Troubleshoot chromogenic OPN5 staining in paraffin sections using the catalog antibody workflow, tissue expression evidence, and controls matched to each specimen.
Which retrieval conditions should I try first for OPN5 paraffin sections?
Start with heat mediated retrieval in EDTA at pH 8.0 for paraffin sections (datasheet A08880-2). The illustrated section used this retrieval before 2 μg/ml primary antibody overnight at 4°C, so keep those conditions together for the first comparison (caption A08880-2). If signal is weak, compare shorter and longer heating in adjacent sections while holding detection constant; document temperature and cooling because retrieval intensity can change signal and morphology (standard IHC practice). Include a no primary control and a retina section enriched in photoreceptor cells to distinguish DAB background from failed epitope access (HPA: High in retinal photoreceptor cells; standard IHC practice).
Could fixation explain inconsistent OPN5 staining between paraffin blocks?
Treat fixation sensitivity of OPN5 as unknown: the selected paraffin section caption does not identify its fixative, and no target specific fixation comparison is supplied (caption A08880-2). Record the actual fixative, fixation duration, section thickness, and processing history for each block before changing the staining procedure (standard IHC practice). Compare adjacent sections from consistently processed blocks using the same EDTA pH 8.0 retrieval and 2 μg/ml primary concentration, then judge signal alongside preserved morphology (datasheet A08880-2; caption A08880-2; standard IHC practice). If staining varies with block history, report the association as an empirical processing effect rather than attributing a molecular mechanism to OPN5 (standard IHC practice).
Where should OPN5 stain, and how should I assess diffuse cytoplasmic colour?
OPN5 is annotated at the cell membrane with 7 transmembrane segments, so assess cell boundaries and relevant processes before treating diffuse colour as specific (UniProt Q6U736 localisation and topology). The tissue atlas reports expression in retinal photoreceptor cells and processes in brain, with supported tissue staining reliability (HPA: High in retinal photoreceptor cells; HPA: expression in brain processes; HPA: Supported). Compare membrane associated signal with adjacent negative cells and a no primary control under identical DAB development (standard IHC practice). Diffuse cytoplasmic signal can reflect intracellular receptor pools or background, so require a reproducible anatomical pattern and independent specificity control before assigning it to OPN5 (standard IHC practice).
Can retrieval differences indicate an OPN5 isoform or modified epitope?
OPN5 has one annotated chain spanning residues 1–354, with 0 annotated isoforms, so an isoform specific staining interpretation is unsupported here (UniProt Q6U736 processing and isoforms). Its 7 membrane spans separate extracellular and cytoplasmic loops; accessibility therefore depends on the antibody's actual epitope and tissue processing (UniProt Q6U736 topology; standard IHC practice). Residue 4 is a glycosylation site, and residue 296 carries a retinylidene modification, but neither establishes which region this antibody recognizes (UniProt Q6U736 modifications). Obtain the immunogen or epitope mapping for A08880-2 before interpreting altered staining after retrieval or claiming modification specific recognition (standard IHC practice).
How should I assess an OPN5 IF result alongside chromogenic tissue staining?
For an IF comparison, pair OPN5 with a marker independently defining the expected cell population; retinal photoreceptor cells are the stated high expression reference (HPA: High in retinal photoreceptor cells). Choose fluorophores after examining unstained tissue autofluorescence, and use separate single stain controls to detect spectral bleed through (standard IF practice). OPN5 crosses the membrane 7 times, with extracellular and cytoplasmic loops, so set permeabilisation according to a documented antibody epitope: a cytoplasmic epitope generally needs membrane access, whereas a surface extracellular epitope may not (UniProt Q6U736 topology; standard IF practice). The paraffin IHC caption supplies no IF validation (caption A08880-2).
What should I check when OPN5 DAB staining appears widespread?
First check no primary and secondary only controls on the same paraffin tissue to identify detection reagent background (standard IHC practice). Quench endogenous peroxidase before HRP and DAB detection, then compare DAB development times across test and control sections (standard IHC practice; caption A08880-2: HRP/DAB detection). The illustrated workflow used 10% goat serum block, 2 μg/ml primary overnight at 4°C, and secondary for 30 minutes at 37°C; retain these as reference conditions when titrating one variable (caption A08880-2). Excess edge or necrotic staining and colour in controls should trigger review of blocking, washing, and tissue integrity before an OPN5 call (standard IHC practice).
How should I quantify OPN5 staining across tissue sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the analysis compartment before counting: score retinal photoreceptor cells separately from neighboring tissue, and specify whether membrane associated signal is required (HPA: High in retinal photoreceptor cells; UniProt Q6U736: cell membrane). For cellular staining, report percent positive cells and an H score based on intensity categories 0–3; count intact cells within a prespecified region (standard IHC practice). For process rich regions, report positive area or process density per mm² rather than forcing a cell count (HPA: expression in brain processes; standard IHC practice). Normalize to eligible cell count or analyzed tissue area, use matched DAB thresholds, and record exclusions for necrosis and edges (standard IHC practice).
How can I distinguish credible OPN5 staining from artefact?
Accept an OPN5 positive pattern when anatomical distribution, compartment, and controls agree: the expected reference is retinal photoreceptor staining with membrane association, while brain processes can also stain (HPA: High in retinal photoreceptor cells; HPA: expression in brain processes; UniProt Q6U736: cell membrane). Review nuclear colour, edge effects, folds, and necrotic deposits as possible artefacts before scoring (standard IHC practice). Test whether colour persists without primary antibody or after peroxidase block; persistence implicates reagent or endogenous enzyme activity rather than the primary antibody (standard IHC practice). The caption demonstrates staining in paraffin embedded human liver cancer tissue without establishing tumour specificity (caption A08880-2).
Boster reagents
Best OPN5 / Opsin-5 IHC Antibodies
Anti-OPN5 antibody A08880-2 has IHC data from human paraffin liver and liver cancer sections (catalog IHC captions) and IF data from a human paraffin liver cancer section (catalog IF caption).
Real IHC data
Anti-OPN5 Antibody ®
Cat # A08880-2
A08880-2 is listed for IHC and IF, with Human, Mouse and Rat reactivity (catalog applications and reactivity). Its IHC captions show human paraffin liver and liver cancer sections; its IF caption shows a human paraffin liver cancer section (catalog image captions).
Which to pick: For tissue IHC, choose A08880-2, a rabbit polyclonal antibody with a listed IHC concentration of 2–5 μg/ml; its IHC images use human paraffin sections, and the fixative is unreported (catalog antibody details; catalog IHC captions). For IF, A08880-2 has a listed concentration of 5 μg/ml and a paraffin-section IF image; ICC validation is unreported (catalog antibody details; catalog IF caption). For cross-species work, A08880-2 lists Human, Mouse and Rat reactivity, while the supplied IHC and IF images show human tissue only (catalog reactivity; catalog image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q6U736 (OPN5_HUMAN, Opsin-5).
- Human Protein Atlas. OPN5 tissue IHC expression (reliability: Supported).
- Human Protein Atlas. OPN5 subcellular location (ICC-IF): Membrane.
- Human Protein Atlas. OPN5 antibody validation summary (1 antibodies).
- Effect of immunization against OPN5 on the reproductive performance in Shan Partridge ducks under different photoperiods. Poultry science 2024 — PMC10964073.
- Expression of novel opsins and intrinsic light responses in the mammalian retinal ganglion cell line RGC-5. Presence of OPN5 in the rat retina. PloS one 2011 — PMC3195719.
- Immunization with OPN5 increased seasonal degradation of reproductive activity in Magang ganders. Poultry science 2025 — PMC11758404.
- PubMed PMID:14623103 — UniProt-cited evidence.
- PubMed PMID:14623098 — UniProt-cited evidence.
- PubMed PMID:12679517 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for OPN5 (Opsin-5). Expected localisation Membrane expected (UniProt); retinal photoreceptors stain (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A08880-2). Validated IHC antibody: A08880-2 at 2-5 μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Retina (Photoreceptor cells); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human liver cancer tissue; fixative not specified (datasheet A08880-2); 2. Fixation: Image fixative and duration unreported (datasheet A08880-2); verify before use.; 3. Sectioning: 4–5 µm sections on charged slides (standard); 4. Deparaffinisation: Xylene, graded ethanol series to water (standard); 5. Antigen retrieval: Heat retrieval: EDTA pH 8.0 (datasheet A08880-2); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A08880-2); 8. Primary antibody: Rabbit anti-OPN5, 2-5 μg/ml (datasheet A08880-2); 9. Primary incubation: Overnight at 4 °C (datasheet A08880-2). Watch for: Fixation: Keep paraffin section fixation consistent (standard IHC practice; not target-specific); Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A08880-2); Caveat: Testis-enriched RNA may mislead tissue choice; retinal staining is high (HPA tissue IHC); Isoform / epitope: No isoforms annotated; epitope side matters across 7 TM spans (UniProt); Regulation: Corneal injury induction, by similarity (UniProt). Evidence: UniProt Q6U736, HPA tissue IHC · Supported, HPA ICC-IF · Membrane, Datasheet A08880-2, PMC10964073, PMC11758404. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/OPN5/immunohistochemistry-ihc-guide.